The present invention relates to methods and devices for modifying eukaryotic cells on functionalized surfaces of a centrifugation apparatus
The conditions during cell culturing have a substantial impact on the phenotypes of the cells and desired or not, cell culturing leads to the manipulation of cells.
Cell culture refers to methods under which eukaryotic cells, especially of mammalian origin, are maintained at appropriate conditions with supply of cell culture medium in a cell incubator or a fermenter. Cell culture conditions vary widely depending on the cell type and the desired application. Variation of cell culture conditions can be utilized for cell expansion, cell differentiation or manufacturing of different phenotypes of the cell type. The most commonly varied factor in culture systems is the cell culture medium, for which a vast number of recipes is known (see for example “Cell Culture Techniques” Humana Press, 1st. Edition, 2011).
Typically culture systems utilize a large amount of medium compared to the mass of the cells to provide a sufficient reservoir for nutrients. In static systems, the medium covering the cells is limiting the gas diffusion to the cells if the cell culture surface itself does not allow gas diffusion. Slow macroscopic convection of the medium results in uncontrolled and uneven supply of nutrients to the cells and may result in different differentiated i.e. manipulated cells.
Culturing large numbers of cells adhered to a surface without the use of carriers or large volume cell suspension is difficult and requires frequent change of the medium. The known static systems for cell culturing are labor-intensive and need clean room conditions during handling the cell cultures, for example media exchange or transfer cells from and into storing devices or adequate incubators for proper cell growth. In dynamic systems for cell culturing like roller fermenters, cells can dislocate from the surface of the fermenter and are suspended in the media. The conditions for growing and supply of nutrients is not uniform for adhered and suspended cells and will result in different differentiated or modified cells. Centrifugation systems for the separation or modification of cells are known.
It is long known to separate cells from a cell mixture into fractions of different cell types with the aid of centrifugal forces in a centrifuge according to their density i.e. their sedimentation velocity. The cell separation is carried out in a specially designed centrifuge, rotor and container (flask) for the cells. For example, whole blood is fractionated or separated by centrifugation into blood plasma (as upper phase), buffy coat (thin layer of leukocytes mixed with platelets in the middle phase), and erythrocytes as lower phase.
The effect of enhanced gravity generated by centrifugation on cells under culturing conditions has been investigated in various publications. Huang et al (2009) disclose in “Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells” in J. Biomed. Here, rBMSCs are first plated on glass coverslips; after 24 h the cells had adhered to the coverslips and the coverslips were transferred to a biocompatible polyethylene culture bag, are incubated with medium and then cultured on a cell centrifuge at 2 g hypergravity for several days. The medium was changed every 3 days during HG/SMG culture.
Gaubin et al. described in Microgravity Sci. Technol. 1991 February; 3(4):246-50 the effects of hypergravity on adherent human cells. Galimberti et al disclose in “Hypergravity speeds up the development of T-lymphocyte motility”, Eur Biophys J, May 1, 2006; 35(5): 393-400 a hypergravity cell culture for 1 to 11 days. Cell culture is performed in flasks which were positioned vertically to the centrifugation axis in the centrifuge. The use of flasks within a centrifuge is furthermore proposed by Versati et al in “Effects of gravity on proliferation and differentiation of adipose tissue-derived stem cells”, J Gravit Physiol, 14(1): P127-128 (2007). Here, a commercial available medium sized centrifuge (MidiCAR) is used to accommodate cell culture flasks to investigate cell growth under hypergravity conditions. Morbidelli et al. investigated in Microgravity Sci. technol (2009) 21:135-140 the effect of hypergravity on endothelial cell function and gene expression. Cell manipulation or cell modification is not disclosed in this publication.
The methods disclosed in these publications are with the exception of hypergravity conditions nearly identical to common cell culturing and involve manual handling steps like medium change. Change of medium i.e. the supply of cells to be cultured with nutrients involves stopping of the centrifugation process, thereby interruption of the enhanced gravitational forces. Manual handling steps are not only laborious and prone to contamination, but also destroy the micro environment of the cells like cell/cell contact or cell/cell interaction. An unaffected micro environment of the cells is important for cell cultivation, e.g. for the activation of lymphocytes or viral or retroviral transduction processes. There is no disclosure in the prior art about the nature of the surface of the flasks or the centrifugation chamber.
It is further known that retroviral transduction of cells can be accelerated by hypergravity, for example described by Tonks et al in Biotechnol Prog. 2005; 21(3): 953-8. With this technique, retrovirus vectors are coated on plates and cells are brought into contact with the virus. In order to promote the contact between target cells and the virus vector, the plate comprising adhered virus and cells are placed into a centrifuge. This requires manual handling steps and the cells are not supplied with medium during centrifugation.
WO 2009/072003 discloses a centrifugation system for cell proliferation. Cell manipulation or cell modification is not disclosed in this publication.
The invention provides a novel device and method for modifying cell populations on functionalized cell modifying surfaces under hypergravity conditions generated by the rotation of a centrifugation chamber. With the device and method of the invention, eukaryotic cells can be modified and/or eukaryotic cells with new or modified features can be generated.